Anorexia in rats infected with the nematode, Nippostrongylus brasiliensis: experimental manipulations.

Nippostrongylus brasiliensis induces a biphasic anorexia in laboratory rats, the first phase coincident with lung invasion (ca day 2) and the second when the worms mature in the intestine (ca day 8). Using the anthelminthic, mebendazole (MBZ), N. brasiliensis infections of the rat were eliminated between the first and second anorexic episodes. This intervention prevented the expression of the second phase of anorexia. Rats exposed to a second infection with N. brasiliensis, 3 weeks after the primary infection, exhibited only a first phase anorexic response which was not influenced by MBZ termination of the primary infection. The lower cumulative food intake and weight gain of all infected rats after 8 days of infection were accompanied by elevated plasma insulin and, in some individuals, by elevated plasma leptin, compared with uninfected controls and previously-infected MBZ-treated rats. Messenger RNA levels for neuropeptide Y were higher in the hypothalamic arcuate nucleus of 8-day infected rats than in recovering MBZ-treated animals. Inoculation of rats with heat-killed N. brasiliensis larvae failed to induce anorexia and did not alter the severity of biphasic anorexia on subsequent injection of viable larvae. The first anorexic episode is therefore dependent upon viable migrating larvae. The second phase of anorexia clearly requires the continuing presence of the parasite beyond the lung phase. Viable migrating larvae are also required to confer 'resistance' to reinfection.

Mode of action of cyclosporin A against Hymenolepis microstoma (Cestoda): relationship between cyclophilin binding and drug-induced damage.

Cyclosporin A (CsA) is a widely investigated experimental anti-parasitic drug whose mode of action remains unresolved. The immunosuppressive action of CsA depends on the drug binding to the intracellular receptor cyclophilin (CyP). This study investigates whether complexing of CsA with parasite CyP is equally essential for its anthelmintic action. The correlation between initial cyclosporin-induced damage in vitro and drug binding to parasite CyP has been examined. CsA and the analogues B-5-49, CsH and CsA-acetate all induced similar damage to the tapeworm Hymenolepis microstoma in vitro in incubations between 2 h and 4 days. The initial foci of drug damage were the parasite surface and mitochondria in the syncytium. In a competitive binding assay only B-5-49 displaced [H3]-CsA from either crude parasite cytosolic CyP or affinity-purified CyP while CsH and CsA-acetate had no effect. These data suggest strongly that cyclosporins act on the surfaces of helminth parasites but that drug action does not involve complex formation with CyP. An alternative drug-binding site must therefore be identified which may lead to the rational design of novel anthelminitic drugs.

Appetite and parasite.

Molecular techniques have dramatically advanced our knowledge of the mechanisms that regulate appetite and body weight, yet human obesity, a significant healthcare problem, still lacks an effective science-based treatment. Infectious agents, including worm parasites, seem capable of overwhelming these regulatory systems. Can the study of parasites extend our understanding of what makes animals feed?

[Ultra-pathological study on the syncytium of Schistosoma mansoni exposed to cyclosporin Ain vitro].

OBJECTIVE: To study the ultra-pathological changes of syncytium of Schistosoma mansoni after cyclosporin A (CsA) treatment. METHODS: MF1 mice were infected with Schistosoma mansoni cercariae. Six weeks later, the adult worms were recovered by portal vein perfusion. After the worms were exposed to CsA of 20 micrograms/ml for 24 h, the drug-induced damage of the worm surface was observed by SEM and TEM. RESULTS: Incubation of male and female schistosomes with 20 micrograms/ml of CsA for 24 h resulted in disruption of the tegument and rupture of the spines. Progressive surface damage and swelling and vacuolization of the tegument led to eventual disruption of the syncytium. CONCLUSION: The antischistosomal action of CsA is direct, the syncytium is the main site for CsA attack.

Mucosal mast cell responses and release of mast cell protease-I in infections of mice with Hymenolepis diminuta and H. microstoma: modulation by cyclosporin A.

The dynamics of intestinal mucosal mast cells and the major mucosal mast cell protease were followed during the course of laboratory infections of mice with Hymenolepis diminuta and H. microstoma. The effects of the drug cyclosporin A (CsA), which is both immunosuppressive and selectively anthelmintic depending upon dose regime, were determined. In H. diminuta infections worm expulsion occurred around day 9 and coincided with peak mastocytosis and peak mMCP-I concentrations in tissues and serum. Immunosuppressive treatment with CsA prevented worm expulsion, permitting some individuals to reach maturity, and abrogated mast cell proliferation and mMCP-I production and release. By contrast, H. microstoma infections persisted for 64 days in spite of a considerable mastocyosis in both intestine and bile duct tissues accompanied by a high level of mMCP-I in tissues and serum. A subimmunosuppressive regime of CsA had only limited effects on worms and mast cell numbers and activity. Together these data shed light on the variable mast cell response to gastrointestinal infections and on the potential significance of parasite location in evasion of mast cell action. Use of CsA reveals the contributions of both T cell-dependent mechanisms, including mast cell proliferation and activation, and T cell-independent events in regulating intestinal helminth infections.

Parasite-induced anorexia: leptin, insulin and corticosterone responses to infection with the nematode, Nippostrongylus brasiliensis.

The nematode parasite, Nippostrongylus brasiliensis, induces a biphasic anorexia in its rat host. The mechanisms, underlying this anorexia and its possible advantages to the host or parasite are unknown. We have investigated the effect of acute (12-24 h) and chronic (2-17 days) infections on plasma concentrations of leptin, insulin and corticosterone, and on hypothalamic expression of neuropeptide Y, galanin and corticotrophin-releasing factor genes. Plasma leptin was elevated in infected rats relative to uninfected ad libitum-fed controls and pair-fed controls in 12 h infections initiated at dark onset and in infections of 2 days' duration. At other times prior to parasite expulsion, plasma leptin in infected and pair-fed rats was lower than that of uninfected ad libitum-fed controls, reflecting the existing state of negative energy balance. Elevated plasma leptin concentrations in infected rats at day 2 post-infection were accompanied by reduced neuropeptide Y gene expression in the hypothalamic arcuate nucleus compared with both ad libitum control and pair-fed animals, and by lowered corticotrophin-releasing factor gene expression in the paraventricular nucleus relative to pair-feds. Twelve hour infections were characterized by a substantial increase in plasma corticosterone that was independent of reduced food intake, and in 12 h infections initiated at dark onset, where plasma leptin was elevated, there was also increased plasma insulin concentration in infected rats. In longer infections, differences between the groups in plasma insulin and corticosterone concentration were only observed at day 4 post-infection. In summary, perturbations to leptin, insulin and corticosterone signals early in infection may have a causative role and might feed back onto hypothalamic gene expression, whereas subsequent changes in these parameters are more likely to be secondary to negative energy balance.

[Chronobiological studies on effect of cyclosporin A (CsA) against adult male worms of Schistosoma mansoni in vitro].

AIM: To study the action mode of cyclosporin A (CsA) against Schistosoma mansoni in vitro. METHODS: MF1 mice were infected with Schistosoma mansoni cercariae for 6 weeks when the adult worms were recovered by portal perfusion. The male worms of S. mansoni recovered were exposed to varying concentrations of CsA at 8, 16, and 24 h in vitro. Drug induced damage to the male worm surface was chrono-biologically observed throughout these experiments by SEM. RESULTS: After the male worms of S. mansoni were incubated with 1 microgram/ml CsA for 8-24 h, the tegument showed swelling of ridges with appearance of holes on their surface and detachment of a part of spines. The above damage of the tegument became more evident in male worms after incubation with 10, 15, 20 micrograms/ml CsA for 8-24 h. Moreover, incubation of male worms with 25 micrograms/ml CsA for 8-24 h resulted in significant deformation and disruption of tegument, rupture of ridges and detachment of spines. The tegumental damage of male worms of S. mansoni was dose- and time-dependent. CONCLUSION: The antischistosomal action of CsA is direct, the schistosome tegument appears to be the main site for CsA attack.

Characterization of calcineurin from Hymenolepis microstoma and H. diminuta and its interaction with cyclosporin A.

The drug cyclosporin A (CsA) exerts its immunosuppressive action by binding to the cytosolic protein, cyclophilin (CyP) and, as a complex, binding to and inhibiting the calcium/calmodulin-dependent serine threonine phosphatase, calcineurin. It is unknown whether a similar mode of action occurs during the drug's antiparasite activity. Calmodulin-binding proteins from the helminth parasites Hymenolepis microstoma and H. diminuta were purified by affinity chromatography, yielding single polypeptide bands of 60000 M(r), according to SDS-PAGE. These proteins were tested for calcineurin activity by the dephosphorylation of the RII peptide (part of the catalytic subunit of cAMP-dependent protein kinase). Both proteins were calcium- and calmodulin-dependent and were inhibited by mammalian cyclophilin complexed with cyclosporin A (IC50 values of 0.75 microgram CyP for H. microstoma and 0.90 microgram CyP for H. diminuta). However, neither of the parasite calcineurins was inhibited by H. microstoma cyclophilin/CsA. These data suggest the anthelmintic mode of action of CsA in these helminth models does not involve the inhibition of a signal transduction pathway requiring interaction with calcineurin.

The antiparasite effects of cyclosporin A: possible drug targets and clinical applications.

1. The immunosuppressive drug cyclosporin A, and some of its nonimmunosuppressive derivatives, are potent inhibitors of a range of parasites of humans. 2. Cyclosporin A and the structurally unrelated immunosuppressant FK506 are known to act on T-lymphocytes as complexes with their binding proteins, cyclophilins and FKBPs, respectively. 3. Cyclophilins and FKBPs have been structurally identified in a number of parasites and, in some instances, are believed to play roles in the antiparasitic actions of these drugs. 4. Nonimmunosuppressive cyclosporins and FK506 derivatives may have clinical potential in certain parasitic diseases, especially malaria and schistosomiasis, and identification of the targets of these drugs in parasites may lead to development of novel chemotherapeutic agents.

Synergistic action of cyclosporin A and polyspecific rabbit anti-sera against murine Schistosoma mansoni.

The efficacy of cyclosporin A (CsA) treatment against Schistosoma mansoni in mice was compared with treatments that included co-administration of one of two anti-sera (infected rabbit serum (IRS) obtained by repeated infection and a worm membrane antigen anti-serum (WSS) obtained by immunization with worm surface supernatants). These two sera recognized a number of worm antigens but differed in precise detail. Administration of CsA alone to mice harbouring mature infections of S. mansoni reduced worm burdens and preferentially targeted female worms. Sera administered alone had no effect on worm burdens. Co-administration of worm membrane antigen anti-serum (WSS) with CsA reduced worm burden significantly compared with drug treatment alone. Male worms were more susceptible to this combined treatment regime. Anti-infection serum (IRS) had a lesser stimulatory activity in combination with CsA which was not statistically different from the effects of CsA alone on worm burdens. The data suggest that CsA-induced surface damage to the parasite may reveal specific antigens that were previously unavailable for host attack.

Hymenolepis diminuta and H. microstoma: uptake of cyclosporin A and drug binding to parasite cyclophilins.

Cyclosporin A (CsA) acts as a powerful immunosuppressant through its binding to the cytosolic isomerase, cyclophilin (CyP), forming a complex which inhibits the phosphatase activity of calcineurin. The drug is also selectively anti-parasitic but its mode of action remains unknown. The mouse tapeworm, Hymenolepis microstoma is sensitive to CsA, but the rat tapeworm, H. diminuta is not susceptible either in rats, mice or in vitro. Using these two tapeworm models, the uptake and binding of CsA were examined in relation to parasite cyclophilins. Uptake and compartmentalization of the drug were markedly different in the two species: H. microstoma takes up more drug than does H. diminuta and sequesters more drug into intracellular compartments. Characterization of cyclophilins using both CsA binding and isomerase activity assays reveals that H. microstoma possesses two cyclophilin isoforms (M(r) 17,700 and 21,400) with isomerase activity that is inhibited by CsA. using identical assays, we have been unable to demonstrate CsA-binding proteins or CsA-sensitive isomerase activity in H. diminuta. These data suggest that the anthelmintic action of CsA relates in some way to the presence and function of parasite cyclophilins.

Anorexia induced by the parasitic nematode, Nippostrongylus brasiliensis: effects on NPY and CRF gene expression in the rat hypothalamus.

Infections of the gastrointestinal nematode, Nippostrongylus brasiliensis, in the laboratory rat result in a characteristic biphasic anorexia which is followed by hyperphagia once the worm burden has been cleared. Despite the importance of parasite-induced anorexia, relatively little is known of the underlying mechanisms. We have investigated the involvement of the central appetite drive in this anorexia by studying the gene expression of two neuropeptides with opposing actions on energy balance, NPY and CRF. Gene expression was assessed by in situ hybridization at 2, 8 and 16 days post-infection (p.i.) in infected rats, in uninfected controls, and in a group with food intake restricted to match that taken voluntarily by the parasitize animals. The sampling intervals corresponded to each of the two phases of maximum anorexia and the period of compensatory hyperphagia. Surprisingly, we found that increases in NPY gene expression in the hypothalamic arcuate nucleus (ARC) accompany anorexia in rats infected with N. brasiliensis; there was a significant relationship between degree of anorexia and induction of NPY mRNA after 8 days of infection. Furthermore, ARC NPY mRNA levels in parasitized animals were similar to those in pair-fed individuals with food intake restricted to match the infected rats. The number of larvae used to establish the infection affected both the degree of anorexia and the level of NPY mRNA at 8 days p.i. in a dose-dependent manner. NPY gene expression remained elevated in infected rats during at least the initial stages of compensatory hyperphagia. This suggests that animals detect a state of energy deficit during the early stages of the infection, yet do not feed, but become hyperphagic coincident with worm loss. The failure of anorectic parasitized animals to feed in response to activation of the NPYergic system makes this a novel system in which to study the regulation of hypothalamic NPY by physiological challenge. There were no significant differences in CRF gene expression between the groups at any of the sampling intervals.

Identification and preliminary characterization of a chitinase gene in the Autographa californica nuclear polyhedrosis virus genome.

A functional chitinase gene (chiA) has been identified in the genome of the Autographa californica nuclear polyhedrosis virus (AcMNPV). It is expressed in the late phase of virus replication in insect cells. High levels of both endo- and exochitinase activity were detected by 12 hr p.i. and remained stable throughout infection. An AcMNPV chiA protein-specific antibody was prepared using recombinant material prepared in bacteria. This was used to demonstrate that a product of approximately 58 kDa was synthesised in virus-infected cells. Immunofluorescence analysis of virus-infected cells showed that most chitinase was located in the cytoplasm. Primer extension analysis of mRNA from AcMNPV-infected cells confirmed that transcription initiated from a baculovirus late start site (TAAG), 14 nucleotides upstream from the putative translation initiation codon. The predicted protein sequence of the AcMNPV chiA shares extensive sequence similarity with chitinases from bacteria and, in particular, the Serratia marcescens chitinase A (60.5% identical residues). Phylogenetic analyses indicate that AcMNPV, or an ancestral baculovirus, acquired the chitinase gene from a bacterium via horizontal gene transfer.

Cyclosporin A: drug treatment in vivo affects the kinetics of [14C]glucose transport in Hymenolepis microstoma in vitro.

The transport of [14C]glucose by Hymenolepis microstoma in vitro following in vivo treatment with cyclosporin A (CsA) was determined over a range of concentrations. For untreated (control) worms glucose uptake showed saturation kinetics with a small diffusion component. Estimates of the maximum velocity of glucose uptake (Vmax) and the affinity of substrate for the glucose transporter (Kt) revealed that untreated 8-day-old worms had a Vmax twice that of 15-day-old worms and that younger worms had a lower Kt. An inverse relationship was demonstrated between log10 worm weight and the rate of uptake of [14C]glucose, reflecting the relatively greater number of glucose transporters due to the larger surface area:volume ratio of smaller worms. Treatment of H. microstoma with CsA in vivo significantly increased the diffusion component of glucose uptake in vitro. Parasites from drug-treated mice had a significantly lower Vmax for glucose uptake than size-matched controls. The affinity of glucose for its transporter in CsA-treated worms (Kt) was not significantly different from size-matched controls. Both juvenile and adult worms underwent transient depletion in total glycogen content after CsA treatment in vivo. The data confirm that CsA treatment in vivo disrupts the functional integrity of the worm tegument, one facet of which is impaired acquisition of glucose.

Human tumor necrosis factor alpha influences rainbow trout Oncorhynchus mykiss leucocyte responses.

Human recombinant tumor necrosis factor alpha (rTNF alpha) and rainbow trout macrophage activating factor (postulated gamma-interferon (gamma-IFN) analogue) synergised to elevate the respiratory burst activity of trout macrophages. This elevated response may parallel phenomena described in mammals where TNF alpha and gamma-IFN commonly synergise. Human rTNF alpha also synergised with mitogenic stimuli to heighten proliferation responses of trout head kidney leucocytes. These data are indicative of a conserved TNF alpha receptor on trout leucocytes (and possibly the TNF alpha molecule itself) and support the notion of an interactive cytokine network regulating immune responses in fish.

Anthelmintic effects of cyclosporin A on protoscoleces and secondary hydatid cysts of Echinococcus granulosus in the mouse.

Cyclosporin A (CsA), employed primarily as an immunosuppressant during the management of organ and graft transplants, exhibits anthelmintic properties. However, its efficacy against tapeworm infections in laboratory models is variable. A preliminary investigation has been undertaken to assess the action of CsA on the establishment and growth of protoscoleces and secondary hydatid cysts of ovine Echinococcus granulosus in mice. Administration of CsA in five consecutive daily doses, beginning 2 days prior to infection, resulted in significant reduction in cyst establishment (measured in terms of cyst masses, cyst numbers and cyst wet weights), when mice were autopsied 20 weeks post-infection. None of these parameters were significantly reduced when the drug was administered 18 weeks post-infection, although wet weight decreased by 42%. Ultrastructural examination of the germinal membrane and laminated layer of late-treated E. granulosus revealed abnormalities in all cysts studied whereas control and early-treated hydatids were normal. A case is made for the consideration of a clinical use for CsA for post-operative control of secondary hydatidosis and its efficacy against hydatid cysts is discussed.

Chitinolytic activities in Heligmosomoides polygyrus and their role in egg hatching.

The occurrence of chitin in the eggshell of Heligmosomoides polygyrus has been determined by histochemical and biochemical techniques. Approximately 5% of the egg dry weight was chitin. Staining with Calcofluor white showed the chitin in the eggshell to be more accessible to the stain after hatching or rupturing of the eggshell. Chitinolytic activity has been detected using fluorescent substrates in extracts of adult males (at low levels), females and eggs. Enzyme activity in situ, within the developing larvae, was visualised with the same substrates. It was localized in discrete granules about 1 micron in diameter which occurred as groups in areas of about 5 microns in diameter, in the posterior third of the larvae. The chitinolytic activity in the eggs increased with the age of the egg and was released into the medium when the eggs hatched. The chitinase activities were very sensitive to inhibition by allosamidin, a specific chitinase inhibitor, with an IC50 for the crude egg extract of 2.2 nM. However, treatment of eggs with 250 microM allosamidin resulted in a slowing but not cessation of egg hatching.